B and T lymphocyte development is driven by V(D)J recombination, a process by which gene segments at the antigen receptor loci are repeatedly rearranged to create a vast repertoire of antigen receptor genes. Because V(D)J recombination entails the cleavage and joining of widely dispersed gene segments many millions of times each day, even a miniscule error rate would still carry considerable risk of translocation. Given this risk, V(D)J recombination is tightly regulated at three main levels: lineage specificity, ordered rearrangement within a given lineage, and allelic exclusion. My lab has been particularly interested in allelic exclusion and how it might be coordinated with ordered rearrangement within a given lineage. Our most recent work, funded by the parent grant ("Co-ordination of recombination and allelic exclusion at Igh and Igk loci") has provided some striking insights into these questions. In brief, we discovered that homologous Ig alleles pair up in a stage-specific manner that parallels the sequential stages of recombination at those loci. IL-7R and STAT5, its downstream signaling component, mediate locus accessibility during Igh rearrangement, promote homologous pairing of Igh alleles, and inhibit Igh/Igk association and Igh locus decontraction. Moreover, we found that interallelic association of Ig loci is mediated by the V(D)J recombinase (i.e., the RAG1 and RAG2 proteins) and the DNA damage checkpoint protein ATM: RAG cleavage on one allele induces repositioning of the other allele to pericentromeric heterochromatin to prevent further cleavage, and this repositioning does not occur in the absence of ATM (Nature Immunology, in press). The involvement of the DNA damage sensing machinery in allelic exclusion is quite surprising and takes my lab into a promising new area of research with direct relevance to oncogenesis. We would now like to expand our existing aims to ask what common mechanisms are involved in coordinating recombination and allelic exclusion of the antigen receptor loci in T cells. Given the unique features of each locus, we expect that these factors and processes could be regulated quite differently in T cells (we already see differences between Igh and Igk, for example, because the former must undergo two rearrangement steps, D to J and then V to DJ). We believe these experiments present a superb opportunity to significantly expand the scope of the parent grant in an eminently logical way. PUBLIC HEALTH REVELANCE: We have been investigating the regulation of ordered, sequential recombination of Igh and Igk loci. Our recent studies have brought to light new regulatory aspects of immunoglobulin rearrangement, that involve STAT5, RAG1 and ATM. In this project we aim to determine whether common mechanisms are involved in coordinating recombination and allelic exclusion of the Tcrb and Tcra loci.